Ingredients for 6 x collagen matrices in a 6-well plate

Method

Pre-chill pipettes, keep collagen on ice

The collagen solidifies above 8ºC

Mix 1.5mL of 10x DMEM with 1.5mL of 10x reconstitution buffer, keep on ice.
Count J2-3T3s and pellet required number in a universal.
Add the 3mL of [1:1, 10x DMEM and 10x reconstitution buffer] and swirl to resuspend the cells, keep on ice

The J2-3T3s seem to survive this somehow

Using chilled pipette, add the 12mL of collagen gently to the cells and tilt to mix, avoiding bubbles as far as possible.

Everything is kept cold to avoid the collagen solidifying

Avoid bubbles

Add 10N NaOH to bring the pH up to 7

Judge the pH visually by the phenol red in the DMEM

Maybe 30–60mL will be necessary

Don’t go too far, use the glacial AcCOOH if you have to, but the more mixing the more bubbles.